The recent discoveries of many bacterial cytoskeleton protein molecules have changed our view on the organization of prokaryotic cells. These molecules form extended filamentous structures to carry out essential cellular functions such as cell shape maintenance, DNA segregation, and cell division. Although being an active area of research, mechanistic understandings of these cellular structures, especially their general impact on the dynamics of cellular chemistry is under-explored. We propose to apply single-molecule imaging techniques (photo-activation single-molecule tracking and photo-activation light microscopy) to studying the dynamics of the intracellular bacterial cytoskeletons. The single-molecule techniques provide unique information due to their ability to localize individual fluorescent molecules with nanometer accuracy, which is particularly valuable for bacterial systems because the small size of most bacteria cells. In this proposal stage, we will focus on: (1) studying the interaction between FtsZ filaments and the potential actin homolog FtsA;(2) characterizing the dynamics of Pbp2 protein in relation to the modeling of the cytoskeleton structures;(3) quantifying the impact of cytoskeleton structures to the mobility of the general population of membrane proteins in bacterial cells. Finally, the proposed project, if successful, will also provide a new experimental technique that is generally useful for studying organized structures in biological cells. PUBLIC HEALTH RELEVANCE: Over the last decade, a common theme emerged from the studies of bacteria of many different species is that there is a great degree of order in the intracellular organizations, despite the small sizes of these cells. The goal of our research is to understand how these ordered structures are produced and how their dynamics are regulated at molecular level with nanometer spatial precision. The knowledge gained would improve our fundamental understandings concerning the biology of prokaryotic life, including many prokaryotic pathogens that are important to human health.